Staphylococcus argenteus from rabbits in Thailand

Abstract Staphylococcus argenteus, a novel species of the genus Staphylococcus or a member of the S. aureus complex, is closely related to S. aureus and is usually misidentified. In this study, the presence of S. argenteus in isolated S. aureus was investigated in 67 rabbits with abscess lesions during 2014–2016. Among 19 S. aureus complex isolates, three were confirmed to be S. argenteus by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, nonribosomal peptide synthetase gene amplification, and multilocus sequence type. All S. aureus complex isolates, including the S. aureus isolates, were examined for their antimicrobial resistance phenotype by disk diffusion and for their resistance genotype by PCR assays. Among the S. argenteus isolates, one was susceptible to all antimicrobial drugs and the other two were resistant to penicillin and doxycycline. In contrast, most S. aureus isolates were resistant to penicillin (37.5%), and gentamicin (12.5%). Moreover, S. aureus isolates harbored the blaZ, mecA, aacA‐aphD, and mrs(A) as well as mutations of gyrA and grlA, but S. argenteus isolates carried solely the blaZ. S. argenteus isolates were investigated for enterotoxin (sea‐sed) and virulence genes by PCR. One isolate carried sea, sec, and sed, whereas the other two isolates carried only sea or sed. No isolate carried seb and see. All three S. argenteus isolates carried hla, hlb, and clfA, followed by pvl, whereas coa, spa (IgG‐binding region), and spa (x region) were not detected in the three isolates. This paper presents the first identification of S. argenteus from rabbits in Thailand. S. argenteus might be pathogenic because the isolates carried virulence genes. Moreover, antimicrobial resistance was observed. Investigations of this new bacterial species should be conducted in other animal species as well as in humans.


| INTRODUC TI ON
The Staphylococcus aureus complex consists of opportunistic pathogens that can cause a wide spectrum of diseases in both humans and animals (Corpa et al., 2009). These pathogens are nonspore-forming, nonmotile, spherical organisms, appearing as grapelike clusters under a microscope. They are facultatively anaerobic, catalase-positive (Foster & Geoghegan, 2015), coagulase-positive and can produce protein A. In rabbits, infection with the S. aureus complex usually results in small dermal lesions; the invasion of subcutaneous tissue and the development of pododermatitis, subcutaneous abscesses, and mastitis. Abscesses in internal organs are sometimes observed, such as in the lungs, liver, and uterus. This gives rise to poor reproductive results, infertility, and death (Corpa et al., 2009;Vancraeynest et al., 2004;Viana et al., 2007).
Recently, a novel coagulase-positive Staphylococcus species, S. argenteus (S. aureus complex), was identified from clinical human and animal sources (Argudín et al., 2016;Chantratita et al., 2016;Schuster et al., 2017;Thaipadungpanit et al., 2015;Tong et al., 2015). Bacterial colonies were characterised by a nonpigmented, creamy white appearance and showed βhemolysis on blood agar. Moreover, the bacteria were shown to be gram-positive cocci in clusters and gave positive results in the catalase and coagulase tests, which are characteristic findings for S. aureus (Tong et al., 2015). Therefore, routine diagnostic analyses can lead to S. argenteus being misidentified as S. aureus. Moreover, identification by molecular methods, such as 16S rRNA sequen cing, cannot differentiate S. argenteus from S. aureus (Tong et al., 2015). Other molecular techniques, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), nonribosomal peptide synthetase (NRPS) gene amplification, and multilocus sequence type (MLST) determination, were thus recommended in many publications as tools for the identification of S. argenteus (Chantratita et al., 2016;Schuster et al., 2017;Zhang et al., 2016). Some sequencing types of S. aureus were previously confirmed to be S. argenteus, such as ST2793, ST1223, and ST2250 (Chantratita et al., 2016;Schuster et al., 2017;Thaipadungpanit et al., 2015;Tong et al., 2015).
The aim of this study was to characterize S. argenteus and S. aureus isolated from rabbits with clinical abscesses.  (Kolecka et al., 2013). As a positive control and calibration reference, 1 ml of Bacterial Test Standard (Bruker Daltonics) was used. The main spectrum was ac-

| NRPS gene amplification
Primer sequences and the PCR protocol for NRPS gene indels were in accordance with those of Zhang et al. (2016). The NRPS gene was amplified in a total reaction volume of 25 μl. The PCR reaction was performed using the thermal cycles (Bio-Rad, California, USA) with initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 40 s and then final extension at 72°C for 10 min. Individual PCR amplicons were purified using the GenepHlow ™ Gel/PCR purification kit (Geneaid, New Taipei, Taiwan) and submitted for sequencing. DNA sequences were analyzed for similarity with the GenBank database.

| MLST
MLST was used to analyze the suspected S. argenteus isolates through the amplification of seven S. aureus housekeeping genes, by a method developed by Enright et al. (2000) at Imperial College London for analyzing a query profile for MLST. Before sequencing, individual PCR amplicons were purified using the GenepHlow ™ Gel/ PCR purification kit (Geneaid). The allelic number queries and sequence types (STs) obtained from trimmed DNA sequencing results for seven genes were determined using the online S. aureus MLST database (https://pubmlst.org/saureus/). The suspected novel alleles or queried allelic profiles of novel STs that did not match the database were submitted to the curator of PubMLST (https://pubmlst. org/saureus/) to check and assign a novel allele or novel ST number.

| Antimicrobial susceptibility testing
Antimicrobial susceptibility testing and interpretation were per-

S. aureus
ATTC ® 25923 was used as the control strain.

| NRPS gene amplification of S. argenteus
The NRPS gene was amplified for eight suspected S. argenteus isolates, as shown in Figure 1. The results showed that three isolates (U19 T10.1, U43 S18.1, and U43 S18.3) have a PCR product of approximately 340 bp, which was correlated with S. argenteus as reported by Zhang et al. (2016). In contrast, the five other suspected isolates had a PCR product of nearly 160 bp, which was reported to correlate with S. aureus (Zhang et al., 2016). The sequences of NRPS amplicons were analyzed for similarity using the GenBank database, which showed that bacterial isolate no. U19 T10.1, U43 S18.1, and U43 S18.  showed close similarity to the S. argenteus group (ST1223, ST2250, ST2854, and ST2198) (Figure 2).

| Antimicrobial resistance genes
In the S. aureus and S. argenteus isolates, the presence of antimicrobial resistance genes was tested. We found that all S. aureus isolates carried blaZ, mecA, aacA-aphD, and mrs(A), but none carried dfrG, tet(K), and cfr, whereas all three S. argenteus isolates carried only the blaZ (isolate no. U43 S18.1 and U43 S18.

| D ISCUSS I ON
Staphylococci are considered the most important veterinary bacterial pathogens because they cause a multiplicity of infections and a wide range of diseases in many host species, including humans and animals (Holmes et al., 2016). This virulent group of pathogens is not only important for livestock, causing conditions such as bovine mastitis or lameness in chickens, but also for causing skin infections resulting in abscesses in pets, such as dogs, cats, and rabbits (Drougka et al., 2016;Goñi et al., 2004;Loncaric et al., 2014;Youn et al., 2014). Pathogenic strains of staphylococci that cause skin infections have been well studied and characterized. Moreover, in farm rabbits, abscesses in the lungs, liver, and uterus lead to poor production, infertility, and death (Corpa et al., 2009 (Thaipadungpanit et al., 2015), but we found the pvl in two S. argenteus isolates in this study. These three S. argenteus isolates, isolated from rabbits, had the ability to cause severe illness in these animals, particularly via the presence of clfA, which usually contributes to abscess formation in rabbits, as previously reported (Malachowa et al., 2016). Although S. argenteus is regionally distributed in animals other than humans, they have been misidentified as S. aureus; this has been suggested in several previous publications (Chantratita et al., 2016;Schuster et al., 2017;Thaipadungpanit et al., 2015;Tong et al., 2015).  (Griggs et al., 2003;Iihara et al., 2006) and codon 80 [TCC (Ser) → TTC (Phe)] in grlA (Aligholi et al., 2011;Iihara et al., 2006). Interestingly, the mutation of grlA at codon 80 found in our study was TCC (Ser) → TTA (Leu), and the mutation of gyrA at codon 88 was GAA (Glu) → GAT (Asp). Comparing the antibiotic resistance pattern, the isolated S. argenteus showed higher susceptibility to antibiotic agents than the isolated S. aureus. However, it is necessary to monitor the development of drug resistance in S. argenteus in the future. From the discovery of S. argenteus in rabbits, further study of its virulence factors, pathogenesis, clinical ma nifestations, antimicrobial resistance, and severity or outcome should be performed to improve our knowledge for treating, controlling, or preventing this novel pathogen in exotic pets.

ACK N OWLED G M ENTS
We thank Prasu-Arthorn Animal Hospital for help with specimen collection. We appreciative Mr. Paul Adams for correcting gram-

CO N FLI C T O F I NTE R E S T
The authors state that there are no conflicts of interest.